The human-pathogenic Yersinia spp. (Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis) are responsible for a range of diseases including diarrhea, mesenteric lymphadenitis, and bubonic plague. These bacteria invade into and colonize the lymphatic organs of humans and a variety of animal hosts. Colonization of a host by Yersinia requires the function of a plasmid-encoded contact-dependent type III secretion system. This type III system translocates a set of toxic proteins known as Yops into host cells. The Yops impair normal host cell signaling functions, resulting in inhibition of phagocytosis, suppression of cytokine synthesis, and induction of apoptosis. The long-term goal of this grant is to understand how Yops modulate host cell signaling functions. The investigators will focus their studies primarily on YopH, a protein tyrosine phosphatase that inhibits phagocytosis, and YopJ, a protein that prevents cytokine synthesis and induces apoptosis. The first specific aim is to carry out a structure/function analysis of an amino-terminal domain in YopH that mediates translocation and substrate recognition. A combination of biophysical and genetic approaches will be used to achieve this goal. The second specific aim is to examine the mechanism of substrate recognition by YopH inside host cells. Animal and cultured cell infection assays will be used to study the behavior of genetically-altered YopH proteins in vivo. The third specific aim is to analyze the interaction of YopJ with host target proteins and to elucidate its mechanism action. Mutant forms of YopJ unable to bind target proteins will be generated and analyzed for biological activity in animal and cultured cell infection assays. The possibility that other Yops modulate the activities of mitogen-activated protein kinases in host cells will also be explored. As type III secretion pathways are important virulence determinants in a large number of bacterial pathogens, and the Yops provide an extremely powerful system to study pathogen interference with host signaling functions, these studies will aid the development of new strategies to combat a variety infectious diseases.